A prokaryotic expression vector for the mutant diphtheria toxin CRM197 was constructed and expressed in Esch-erichia coli cells.Anti-CRM197 antibody concentrations were detected in serum samples of healthy volunteers.The crm 197 gene was codon-optimized in E.coli and cloned into the plasmid pET28a(+)under optimized expression conditions.CRM197 was purified using Ni-NTA spin columns and ion exchange chromatography,and confirmed by western blot analysis.The puri-fied CRM197 was used to detect specific anti-CRM197 antibody levels in serum samples of different age groups.The results showed that soluble codon-optimized CRM197 was successfully expressed under optimized expression conditions.The purity of CRM197 was more than 95％,as determined with Ni-NTA spin columns and ion exchange chromatography,consistent with the single specific bands obtained by western blot analysis and detection of serum levels of the anti-CRM197 antibody.Collec-tively,these results confirmed that the proposed expression strategy achieved high-yield production of soluble CRM197,al-though high levels in human serum may affect evaluation of immune interactions with glycan-CRM197 conjugates for applica-tion as a diagnostic antigen.The diphtheria mutant toxin CRM197 is used in many conjugate vaccines.The synthetic crm 197 gene with codon optimization in pET28a was transformed into E.coli Origami B(DE3)cells.CRM197 was induced by isopro-pyl β-d-1-thiogalactopyranoside and high level accumulation of soluble CRM197 was purified using Ni-NTA spin columns and ion exchange chromatography.The purity of the final prepara-tion reached 95％.CRM197 was used to detect the concentra-tions of the anti-CRM197 antibody in serum samples of healthy volunteers of different ages.The proposed expression strategy yielded high production of CRM197,which could interfere with evaluations of induced immune interactions by glycan-CRM197 conjugates and prohibit application as a diagnostic antigen.

Objective:To investigate the effects of Curcumol on the malignant biological characteristics of acute myeloid leukemia (AML)cells and its molecular mechanism,and to provide theoretical and experimental evidence for the anti-leukemia treatment of traditional Chinese medicine.Methods:After the AML cell lines HL-60 and KG-1 cells were treated different concentrations of with Curcumol.The proliferation activity of cells was detected by CCK-8 method,and the expression changes of apoptotic proteins and PI3 K/AKT signaling pathway proteins were detected by Western blot. Real-time quantitative fluorescence polymerase chain reaction (RT-qPCR ) was used to detect the expression of Caspase family mRNA.Results:Curcumol could inhibit the proliferation and induce apoptosis of HL-60 and KG-1 cells,promote apoptosis by up-regulating the expression of Bax and down-regulating the expression of Bcl-2 protein (P<0.05).When Curcumol interferes with HL-60 and KG-1 cells,it can also induce programmed cell death of AML by inhibiting PI3 K/AKT signaling pathway.In addition,after the intervention of Curcumol,the expression of Caspase 3,Caspase 6,Caspase 8 and Caspase 9 were up-regulated in HL-60 cells (P<0.05 ),the expression of Caspase 3,Caspase 8 and Caspase 9 were significantly up-regulated in KG-1 cells (P<0.01),while the expression of Caspase 6 was weakly affected (P<0.05 ),but low concentration of Curcumol (<60 μg/ml)had no effect on the expression of Caspase 6 in KG-1 cells (P>0.05).Conclusion:Curcumol may mediate the programmed death of AML cells by inhibiting the PI3K/AKT signaling pathway,affecting the expression of Bcl-2 family proteins,and promoting the activation of core members of Caspase family,so as to play an anti-leukemia role.

AIM To evaluate the quality of Yanyangke Mixture.METHODS The HPLC fingerprints were established,after which cluster analysis,principal component analysis and partial least squares discriminant analysis were performed.The contents of liquiritin,rosmarinic acid,sheganoside,irisgenin,honokiol,monoammonium glycyrrhizinate,irisflorentin,isoliquiritin and magnolol were determined,the analysis was performed on a 35 ℃ thermostatic Agilent ZORBAX SB-C18 column(5 μm,250 mmx4.6 mm),with the mobile phase comprising of 0.1％phosphoric acid-acetonitrile flowing at 1 mL/min in a gradient elution manner,and multi-wavelength detection was adopted.RESULTS There were ten common peaks in the fingerprints for twelve batches of samples with the similarities of more than 0.9.Various batches of samples were clustered into three types,three principal components displayed the acumulative variance contribution rate of 87.448％,peaks 5、14(honokiol),3(liquiritin),11(monoammonium glycyrrhizinate)and 15(asarinin)were quality markers.Nine constituents showed good linear relationships within their own ranges(r＞0.999 0),whose average recoveries were 98.5％-103.6％with the RSDs of 0.92％-1.7％.CONCLUSION This stable and reliable method can provide a basis for the quality control of Yanyangke Mixture.

Shiwei-Ruxiang-capsule (SWRXC) is a classic formulation widely used in the treatment of rheumatoid arthritis (RA). The study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) serum untargeted metabolomics and high-throughput 16S rRNA gene sequencing association analysis to elucidate the mechanism of action of SWRXC for the treatment of Freund's complete adjuvant-induced RA. The results showed that SWRXC significantly improved symptoms and reduced serum cytokine levels in RA rats. Based on LC-MS/MS technology, metabolomics identified tryptophan metabolism, nucleotide metabolism and purine metabolism as the most relevant pathways for treatment. In addition, 16S rRNA sequencing results showed that SWRXC could ameliorate RA-induced intestinal microbial oncogenesis in rats. In conclusion, SWRXC can improve the morphology and structure of RA joint tissues, reduce serum factor levels, and may play a role in improving RA by modulating related metabolic pathways such as tryptophan metabolism, nucleotide metabolism and purine metabolism, and altering the composition of intestinal flora. Animal protocols were approved by the Animal Ethics Committee of Qinghai Normal University (No. 2021041203).

The aim of this paper is to explore the key anti-fatigue active components in the saponin-like composition of American ginseng. The anti-fatigue activity of western ginseng samples was evaluated using a zebrafish model; metabolomics techniques were used to identify the main saponins in western ginseng from different origins; the active substances and relevant targets of the anti-fatigue effect of western ginseng were initially screened by constructing a PPI protein interaction network between western ginseng saponins and disease targets, and the key active ingredients were screened using a molecular docking method; finally, the anti-fatigue activity of the key active ingredients was evaluated using a zebrafish, animal experiment was approved by the Ethics Committee of Shandong Academy of Medical Sciences (SYXK20220005). The anti-fatigue activity of the key active ingredients was evaluated using a zebrafish model. The results of the zebrafish activity evaluation showed that there were significant differences in the activities of the western ginseng samples from the two origins, and a total of 10 different saponins were identified as possibly related to the anti-fatigue activity after further metabolomic testing and pattern discrimination. The core anti-fatigue targets were screened with the help of component-disease target PPI, combined with pharmacophore-like parameters and molecular docking techniques, and pseudoginsenoside F11 was found to have good binding activity to five of the targets. Finally, the zebrafish model revealed that pseudoginsenoside F11 exhibited significant anti-fatigue activity. This study used metabolomics and zebrafish model to screen the key active substances of pseudoginsenoside F11 for its anti-fatigue activity, which will provide a reference for further research on the anti-fatigue of pseudoginsenosides.

OBJECTIVE: Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research. In this report, two distinguished phenotypic isolates (CJ1Z, flhA mutant strain, lawn; CJ2S, flhA complemented strain, normal colony) appeared during laboratory passages for NCTC11168. METHODS: Phenotypic assessments, including motility plates, transmission electron microscopy, biofilm formation assay, autoagglutination assay, and genome re-sequencing for these two isolates (CJ1Z, flhA mutant strain; CJ2S, flhA complemented strain) were carried out in this study. RESULTS: Transmission electron microscopy revealed that the flagellum was lost in CJ1Z. Phenotypic assessments and genome sequencing of the two isolates were performed in this study. The capacity for biofilm formation, colony auto-agglutination, and isolate motility was reduced in the mutant CJ1Z. Comparative genomic analysis indicated a unique native nucleotide insertion in flhA (nt, 2154) that caused the I719Y and I720Y mutations and early truncation in flhA. CONCLUSION FlhA has been found to influence the expression of flagella in C. jejuni. To the best of our knowledge, this is the first study to describe the function of the C-terminal of this protein.
Campylobacter jejuni/genetics* ; Bacterial Proteins/metabolism* ; Mutation ; Biological Variation, Population

OBJECTIVE: To monitor the changes of voriconazole minimum concentration(C_min) in patients with hematological diseases, and evaluate the factors influencing and adverse reactions of voriconazole clearance in patients with hematological diseases, so as to provide a theoretical basis for reasonable clinical use of voriconazole. METHODS: 136 patients with hematological diseases who used voriconazole in Wuhan NO.1 Hospital from May 2018 to December 2019 were selected. The correlation between C-reactive protein, albumin, creatinine and voriconazole C_min were analyzed, and the changes of voriconazole C_min after glucocorticoid treatment was also detected. In addition, stratified analysis was used to explore the adverse events of voriconazole. RESULTS: Among 136 patients, 77 were male (56.62%) and 59 were female (43.38%). There were positive correlations between voriconazole C_min and C-reactive protein and creatinine levels (r=0.277, r=0.208), while voriconazole C_min was negatively correlated with albumin level (r=-2.673). Voriconazole C_min in patients treated with glucocorticoid was decreased significantly (P<0.05). In addition, sratified analysis of voriconazole C_min showed that compared with voriconazole C_min 1.0-5.0 mg/L group, the incidence of adverse reactions of visual impairment in voriconazole C_min> 5.0 mg/L group was increased (χ²=4.318, P=0.038). CONCLUSION The levels of C-reactive protein, albumin and creatinine are closely related to the voriconazole C_min, which indicate that inflammation and hyponutrition may prevent the clearance of voriconazole in patients with hematological diseases. It is necessary to monitor the voriconazole C_min of patients with hematological diseases, and adjust the dosage in time to reduce adverse reactions.
Humans ; Male ; Female ; Voriconazole/therapeutic use* ; Antifungal Agents/therapeutic use* ; C-Reactive Protein ; Creatinine ; Glucocorticoids ; Retrospective Studies ; Drug Monitoring ; Hematologic Diseases

OBJECTIVE: To analyze the important effect of 3D printing personalized lumbar support on lumbar pain and lumbar function in patients with lumbar disc herniation. METHODS: From October 2018 to May 2021, 60 patients initially diagnosed with lumbar disc herniation were selected and divided into an observation group and a control group, with 30 patients in each group. Among them, there were 18 males and 12 females in the observation group;the age ranged from 24 to 56 years old, with an average of (45.23±6.07) years old. The course of disease ranged from 1 to 24 months, with an average of(6.25±0.82) months, and rehabilitation treatment was carried out by wearing 3D printed personalized lumbar support. There were 19 males and 11 females in the control group;the age ranged from 25 to 57 years old, with an average of (42.78±7.58) years old. The course of disease ranged from 1 to 24 months, with an average of (6.72±1.36) months, and rehabilitation treatment is carried out by wearing traditional lumbar protective equipment. The Japanese Orthopaedic Association (JOA) scores, lumbar Oswestry dysfunction index (ODI) and visual analogue scale (VAS) were evaluated and compared between the two groups before and 1 course after treatment (3 weeks). RESULTS: There was no statistically significant difference in JOA, ODI, and VAS between two groups before treatment (P>0.05). After one course of treatment (3 weeks), JOA scores of both groups was increased compared to before treatment (P<0.05), while ODI and VAS decreased compared to before treatment (P<0.05). After treatment, JOA score of observation group was higher than that of control group (P<0.05), while ODI and VAS scores were lower than those of control group. No adverse events occurred in both groups. CONCLUSION The application of 3D printing personalized lumbar support can effectively alleviate the pain of patients with lumbar disc herniation and improve their lumbar function of patients.
Female ; Male ; Humans ; Young Adult ; Adult ; Middle Aged ; Intervertebral Disc Displacement/surgery* ; Printing, Three-Dimensional ; Technology ; Orthopedics ; Low Back Pain

Objective: To analyze and compare therapy responses, outcomes, and incidence of severe hematologic adverse events of flumatinib and imatinib in patients newly diagnosed with chronic phase chronic myeloid leukemia (CML) . Methods: Data of patients with chronic phase CML diagnosed between January 2006 and November 2022 from 76 centers, aged ≥18 years, and received initial flumatinib or imatinib therapy within 6 months after diagnosis in China were retrospectively interrogated. Propensity score matching (PSM) analysis was performed to reduce the bias of the initial TKI selection, and the therapy responses and outcomes of patients receiving initial flumatinib or imatinib therapy were compared. Results: A total of 4 833 adult patients with CML receiving initial imatinib (n=4 380) or flumatinib (n=453) therapy were included in the study. In the imatinib cohort, the median follow-up time was 54 [interquartile range (IQR), 31-85] months, and the 7-year cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) were 95.2%, 88.4%, 78.3%, and 63.0%, respectively. The 7-year FFS, PFS, and OS rates were 71.8%, 93.0%, and 96.9%, respectively. With the median follow-up of 18 (IQR, 13-25) months in the flumatinib cohort, the 2-year cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) were 95.4%, 86.5%, 58.4%, and 46.6%, respectively. The 2-year FFS, PFS, and OS rates were 80.1%, 95.0%, and 99.5%, respectively. The PSM analysis indicated that patients receiving initial flumatinib therapy had significantly higher cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) and higher probabilities of FFS than those receiving the initial imatinib therapy (all P<0.001), whereas the PFS (P=0.230) and OS (P=0.268) were comparable between the two cohorts. The incidence of severe hematologic adverse events (grade≥Ⅲ) was comparable in the two cohorts. Conclusion: Patients receiving initial flumatinib therapy had higher cumulative incidences of therapy responses and higher probability of FFS than those receiving initial imatinib therapy, whereas the incidence of severe hematologic adverse events was comparable between the two cohorts.
Adult ; Humans ; Adolescent ; Imatinib Mesylate/adverse effects* ; Incidence ; Antineoplastic Agents/adverse effects* ; Retrospective Studies ; Pyrimidines/adverse effects* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy* ; Treatment Outcome ; Benzamides/adverse effects* ; Leukemia, Myeloid, Chronic-Phase/drug therapy* ; Aminopyridines/therapeutic use* ; Protein Kinase Inhibitors/therapeutic use*

Diabetic retinopathy(DR)is a prevalent microvascular complication of diabetes and the leading cause of blindness and severe visual impairment in adults.The high levels of glucose trigger multiple intracellular oxidative stress pathways,such as POLDIP2,resulting in excessive reactive oxygen species(ROS)pro-duction and increased expression of vascular cell adhesion molecule-1(VCAM-1),hypoxia-inducible factor 1α(HIF-1α),and vascular endothelial growth factor(VEGF),causing microvascular dysfunction.Dihydromyricetin(DMY)is a natural flavonoid small molecule antioxidant.However,it exhibits poor solubility in physiological environments,has a short half-life in vivo,and has low oral bioavailability.In this study,we present,for the first time,the synthesis of ultra-small Fe-DMY nano-coordinated polymer particles(Fe-DMY NCPs),formed by combining DMY with low-toxicity iron ions.In vitro and in vivo experiments confirm that Fe-DMY NCPs alleviate oxidative stress-induced damage to vascular endo-thelial cells by high glucose,scavenge excess ROS,and improve pathological features of DR,such as retinal vascular leakage and neovascularization.Mechanistic validation indicates that Fe-DMY NCPs can inhibit the activation of the Poldip2-Nox4-H2O2 signaling pathway and downregulate vital vascular function indicators such as VCAM-1,HIF-1α,and VEGF.These findings suggest that Fe-DMY NCPs could serve as a safe and effective antioxidant and microangio-protective agent,with the potential as a novel multimeric drug for DR therapy.